The poxvirus vaccinia, which is the live vaccine virus used in the eradication of smallpox, is a large double stranded DNA containing virus that replicates in the has been to understand genome using a genetic vaccinia has uncovered be critical for proper includes vaccinia gene increased steady state cytoplasm of infected cells. Our long-range goal the functional organization of the vaccinia approach. Our ongoing genetic analysis of a network of essential viral genes which seem to control of late viral transcription. The network A18R, which when mutated leads to synthesis of levels of late viral RNA, and gene G2R, which when mutated leads to decreased synthesis of late viral proteins. In this application, we propose to extend the analysis of this network and continue our efforts to develop directed in vitro methods for construction of conditionally lethal mutations in essential vaccinia genes. The specific aims are: 1) optimize and characterize a virus specific in vitro transcription system, 2) using biochemical protocols and appropriate mutants, characterize the product of the A18R gene and determine its role in infection, 3) using biochemical protocols and appropriate mutants, characterize the product of the G2R gene and determine its role in infection, 4) using genetic protocols, determine .the relationship between genes A18R and G2R, and seek additional genes involved in the network, and 5) test alanine scanning mutagenesis coupled with transient dominant selection as a method for directed in vitro construction of conditionally lethal mutants of vaccinia.